An important feature of mammals is the sexually dimorphic reproductive tracts, which include external and internal sex organs. Abnormal development of the genital tubercle (GT), the common embryonic precursor of both male and female external genitalia, frequently results in birth defects including ambiguous genitalia and hypospadias but the underlying molecular basis is poorly understood. This proposal focuses on formation of the external genitalia, a significantly understudied area of embryology. Based on our preliminary data, we hypothesize that, in addition to the gonad-derived sex hormones such as androgens, Wnts expressed from the GT epithelium function as the permissive signal and; following the Wnt and androgen stimulation the gender-specific genomic activity of GT mesenchyme leads to formation of gender-specific external genital structures. We have designed three specific aims to test this hypothesis: 1) to examine whether Wnt4 is the permissive signal for dimorphic GT differentiation; 2) to determine whether sex hormones reprogram the GT- specific landscape of transcription enhancers to control dimorphic gene expression; and 3) to identify critical GT-specific downstream effectors of sex hormones that induce dimorphic GT differentiation to form penis in males and clitoris in females. We have developed and validated a suite of innovative genetic and genomic tools in order to achieve these specific aims. Findings from the proposed studies are expected to improve our understanding of sexual dimorphism in mammals, and to shed new light on the pathogenesis of common birth defects including ambiguous genitalia and hypospadias.